Gb virus b based replicons and replicon enhanced cells

ABSTRACT

The present invention features GBV-B replicons and replicon enhanced cells. A GBV-B replicon is an RNA molecule able to autonomously replicate in a cultured cell and produce detectable levels of one or more GBV-B proteins. GBV-B replicon enhanced cells are cells having an increased ability to maintain a GBV-B replicon.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to provisional application U.S.Ser. No. 60/386,655, filed Jun. 6, 2002, and provisional applicationU.S. Ser. No. 60/348,573, filed Jan. 15, 2002, hereby incorporated byreference herein.

BACKGROUND OF THE INVENTION

The references cited throughout the present application are not admittedto be prior art to the claimed invention.

It is estimated that about 3% of the world's population is infected withthe hepatitis C virus (HCV). (Wasley et al., Semin. Liver Dis. 20:1-16,2000.) HCV exposure results in an overt acute disease in a smallpercentage of cases, while in most instances the virus establishes achronic infection causing liver inflammation and slowly progresses intoliver failure and cirrhosis. (Strader et al., ILAR J. 42:107-116, 2001.)Epidemiological surveys indicate an important role for HCV in the onsetof hepatocellular carcinoma. (Strader et al., ILAR J. 42:107-116, 2001).

Investigating the effects of HCV and antiviral compounds is complicatedby the absence of a small animal model. HCV infects human andchimpanzees, but does not infect small animals such as mice and rats.

The GB virus B (GBV-B) can infect different new world monkeys such astamarins and owl monkeys. (Bukh et al., Journal of Medical Virology65:694-697, 2001.) GBV-B has been proposed as a surrogate model forstudying HCV and the effects of antiviral compounds. (Traboni,International Publication Number WO/73466, International PublicationDate 7 Dec. 2000, Bukh et al., International Publication NumberWO/75337, International Publication Date 14 Dec. 2000).

The hypothesis of deriving information useful for research of anti-HCVdrugs from experiments with GBV-B in tamarins has been supported by dataconcerning enzymatic activity of viral proteins and the role ofuntranslated regions, as well as the identification of in vivoinfectious cDNA and the establishment of a cell-based infection system.(See for example, Beames et al., J. Virol. 74:11764-11772, 2000, Traboniet al., The GB viruses: a comprehensive survey. In S. G. Pandalai (ed.),Recent Research Developments in Virology, part III. Transworld ResearchNetwork, 1999.)

The similarity between HCV and GBV-B genome organization was underlinedsince GBV-B was discovered in 1995. (Muerhoff et al., J. Virol.69:5621-5630, 1995.) Early experimental demonstration of the similarityat the functional level came from the enzymatic activity of NS3protease. (Scarselli et al., J. Virol. 71:49854989, 1997.) Subsequentanalyses have been performed looking at different HCV and GBV-B regions.

Studies performed examining polyprotein processing and the functionalrelationship between the HCV and GBV-B NS3 proteins indicate overlappingspecificity, and a virus-specific NS4A cofactor requirement. (Butkiewiczet al., J. Virol. 74:4291-4301, 2000, Sbardellati et al., J. Gen. Virol.81 Pt 9:2183-2188, 2000.)

The helicase and NTP-ase activity associated with the C-terminal domainof GBV-B NS3 protein has been reported as comparable to those of HCV.(Zhong et al., Virology. 261:216-226, 1999.) The RNA-dependent RNApolymerase activity encoded by a truncated form of GBV-B NS5B and HCVNS5B showed similarities. (Zhong et al., J. Viral Hepat. 7:335-342,2000).

The HCV and GBV-B 5′ and 3′ untranslated regions play an important rolein the initiation of the replication process via interactions with viralproteins such as helicase and RNA-dependent RNA polymerase. The HCV andGBV-B 5′ and 3′ untranslated regions contain common features. Theinternal ribosome entry site containing 5′-UTR of GBV-B shows bothstructural and functional similarity to that of HCV. (Grace et al., J.Gen. Virol. 80:2337-2341, 1999, Rijnbrand et al., J. Virol. 74:773-783,2000, Rijnbrand et al., Rna. 7:585-597, 2001.) The 3′end of the GBV-B3′UTR is arranged in a similar secondary structure to HCV and isimportant for replication and in vivo infectivity. (Bukh et al.,Virology 262:470-478, 1999, Sbardellati et al., Journal of Virology73:10546-10550, 1999, Sbardellati et al., J. Gen. Virol. 82:2437-2448,2001.)

SUMMARY OF THE INVENTION

The present invention features GBV-B replicons and replicon enhancedcells. A GBV-B replicon is an RNA molecule able to autonomouslyreplicate in a cultured cell and produce detectable levels of one ormore GBV-B proteins. GBV-B replicon enhanced cells are cells having anincreased ability to maintain a GBV-B replicon.

Functional GBV-B genomic and subgenomic replicons can be obtained basedon GBV-B sequences such as those provided in SEQ ID NO: 1 and SEQ. ID.NO. 2. SEQ. ID. NO. 1 provides a bicistronic subgenomic repliconsequence illustrated herein as able to replicate in a cell. SEQ. ID. NO.2 provides the cDNA sequence of a genomic GBV-B replicon infectious intamarins.

GBV-B replicon enhanced cells can be produced by selecting for a cellable to maintain a GBV-B replicon and curing the cell of the replicon.The replicon enhanced cell has an increased ability to maintain areplicon upon subsequent transfection. The replicon used in thesubsequent transfection can be different from the replicon used toproduce the replicon enhanced cell. For example, a bicistronic GBV-Breplicon with a selection/reporter sequence can be used to obtain theenhanced cell and a second replicon without such a selection/reportersequence, including a full-length infectious replicon, can be introducedinto the replicon enhanced cell.

Thus, a first aspect of the present invention describes a GBV-B repliconcapable of replication in a cell comprising the following regions:

-   -   a GBV-B 5′ UTR substantially similar to bases 1-445 of SEQ. ID.        NO. 1;    -   a selection or reporter sequence functionally coupled to the        GBV-B 5′ UTR;    -   an internal ribosome entry site;    -   a NS3-NS5B sequence substantially similar to bases 1938-7709 of        SEQ. ID. NO. 1 functionally coupled to the internal ribosome        entry site and an AUG translation initiation codon; and    -   a GBV-B 3′ UTR substantially similar to bases 7710-8069 of SEQ.        ID. NO. 1.

Reference to “comprising the following regions” indicates that theprovided regions are present and additional regions may also be present.The additional regions are preferably located in a position between the5′ GBV-B UTR and GBV-B 3′ UTR. However, the additional region can be,for example, an additional cistron located 5′ of the 5′ GBV-B UTR.

Preferred additional regions are GBV-B structural region(s) and theGBV-B NS2 region. Additional regions can be of different sizes such as apartial core region or a complete structural GBV-B region and can beprovided together in one region. For example, the NS2 region can beprovided at the 3′ end of a structural region.

Additional regions can be present in different replicon locations.Examples of different locations include providing a structural regionand/or NS2 region in a cistron containing the GBV-B 5′ UTR and providinga structural region and/or NS2 region in a cistron comprising NS3-NSSB.

Reference to “functionally coupled” indicates the ability of a firstnucleotide sequence to mediate an effect on a second nucleotidesequence. Functionally coupled does not require that the coupledsequences be adjacent to each other. A GBV-B 5′-UTR and an internalribosome entry site facilitates ribosome binding and/or translation ofthe sequences to which they are coupled. The GBV-B 3′ UTR is importantfor replicon replication.

Another aspect of the present invention describes an expression vectorcomprising a promoter transcriptionally coupled to a nucleotide sequencecoding for a GBV-B replicon described herein.

Another aspect of the present invention describes a GBV-B replicon madeby a process comprising the steps of transfecting a cell with a GBV-Breplicon and isolating the replicon.

Another aspect of the present invention describes a method of making asecond GBV-B replicon from a first GBV replicon comprising the steps of:(a) transfecting a cell with the first replicon; (b) isolating areplicon from the transfected cell; (c) determining the nucleotidesequence of the replicon from the transfected cell; and (d) producingthe second GBV-B replicon, wherein the second replicon contains thefirst replicon sequence with one or more alterations corresponding tothe transfected cell replicon sequence. Preferably, the second repliconhas the same sequence as the transfected cell replicon sequence.

Another aspect of the present invention describes a method of measuringthe ability of a compound to affect GBV-B replicon activity. The methodinvolves providing the compound to a cell containing a GBV-B repliconand measuring the ability of the compound to affect one or more repliconactivities as a measure of the effect on GBV-B activity.

Another aspect of the present invention describes a GBV-B repliconenhanced cell wherein the cell has a maintenance and activity efficiencyof at least 25% when transfected with a GBV-B replicon of SEQ ID. NO. 1by the Electroporation Method.

Reference to the “Electroporation Method” indicates the transfectiontechniques described in Example 1 infra. The replicon enhanced cellsneed not be produced from a particular cell type or by a particulartechnique, but rather has as a property a maintenance and activityefficiency of at least 25% upon transfection of the GBV-B replicon ofSEQ. ID. NO. 1 using the Electroporation Method.

A maintenance and activity efficiency of at least 25% indicates that atleast 25% of the cells used in the Electroporation Method maintainfunctional replicons. In different embodiments the maintenance andactivity efficiency is at least 35%, at least 50%, or at least 75%.

Preferred replicon enhanced cells are Huh7 or Hep3B derived cells.“Derived cells” are cells produced starting with a particular cell(e.g., Huh7 or Hep3B) and selecting, introducing or producing one ormore phenotypic or genotypic modifications.

Another aspect of the present invention describes a method of making aGBV-B replicon enhanced cell. The method involves the steps of: (a)introducing and maintaining a GBV-B replicon into a cell and (b) curingthe cell of the replicon.

Another aspect of the present invention describes a GBV-B repliconenhanced cell made by a process comprising the steps of: (a) introducingand maintaining a GBV-B replicon into a cell and (b) curing the cell ofthe replicon.

Another aspect of the present invention describes a method of making aGBV-B replicon enhanced cell comprising a GBV-B replicon. The methodinvolves producing a replicon enhanced cell and introducing andmaintaining a GBV-B replicon in the cell.

Another aspect of the present invention describes a GBV-B repliconenhanced cell containing a GBV-B replicon made by a process involvingproducing a replicon enhanced cell and introducing and maintaining theGBV-B replicon in the cell.

Another aspect of the present invention describes a method of measuringthe ability of a compound to affect GBV-B replicon activity using aGBV-B replicon enhanced cell comprising a GBV-B replicon. The methodinvolves providing a compound to the cell and measuring the ability ofthe compound to affect one or more replicon activities as a measure ofthe effect on GBV-B replicon activity.

Another aspect of the present invention describes a method of producingan infectious GBV-B virion. The method comprises the steps of growing areplicon enhanced cell containing a replicon encoding a GB V-B virion toproduce the GBV-B virion.

Another aspect of the present invention describes a method of infectingan animal with a GBV-B virion. The method involves producing the virionand providing the virion to an animal.

Another aspect of the present invention describes a method for producinga chimeric GBV-B/HCV replicon. The method involves the step of replacingone or more GBV-B regions or portion thereof present in a replicondescribed herein with the corresponding region from HCV.

Other features and advantages of the present invention are apparent fromthe additional descriptions provided herein including the differentexamples. The provided examples illustrate different components andmethodology useful in practicing the present invention. The examples donot limit the claimed invention. Based on the present disclosure theskilled artisan can identify and employ other components and methodologyuseful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F provide the neo-RepD replicon sequence (SEQ. ID. NO. 1).Nucleotide number 1 is the first of GBV-B genome. Core region is incapital letters. The approximate location of the GBV-B regions areprovided as follows:

-   1-445: GBV-B 5′ non-translated region, drives translation of the    core-neo fusion protein;-   446-1315 (including stop codon): core-neo fusion protein, selectable    marker;-   1324-1934: Internal ribosome entry site of the encephalomyocarditis    virus, drives translation of the GBV-B NS region;-   1935-7709: GBV-B polyprotein from non-structural protein 3 to    non-structural protein 5B, including an AUG start codon;-   1938-3797 (putative): Non-structural protein 3 (NS3), NS3    protease/helicase;-   3798 (putative)-3962: Non-structural protein 4A (NS4A), NS3 protease    cofactor;-   3963-4706 (putative): Non-structural protein 4B (NS4B);-   4707 (putative)-5939: Non-structural protein 5A (NS5A);-   5940-7709 (excluding stop codon): Non-structural protein 5B (NS5B);    GBV-B RNA-dependent RNA polymerase; and-   7710-8069: GBV-B 3′ non-translated region.

FIGS. 2A-2G illustrate the cDNA (SEQ. ID. NO. 2) for a full-length GBV-Breplicon sequence infectious in tamarins (Sbardellati et al., J. Gen.Virol. 82:2437-2448, 2001). The approximate location of the GBV-Bregions are provided as follows:

-   1-445: GBV-B 5′ non-translated region, drives translation of the    GBV-B polyprotein;-   446-9037: GBV-B polyprotein from core protein to non-structural    protein 5B;-   446-919 (putative): structural protein core, nucleocapsid protein;-   920 (putative)-1489 (putative): structural protein E1, envelope    protein;-   1490 (putative)-2641 (putative):structural protein E2, envelope    protein;-   2642 (putative)-3265: Non-structural protein 2 (NS2);-   3266-5125 (putative): Non-structural protein 3 (NS3), GBV-B NS3    protease/helicase;-   5126 (putative)-5289: Non-structural protein 4A (NS4A), NS3 protease    cofactor;-   5290-6034 (putative): Non-structural protein 4B (NS4B);-   6035 (putative)-7267: Non-structural protein 5A (NSSA);-   7268-9037 (excluding stop codon): Non-structural protein 5B (NS5B);    GBV-B RNA-dependent RNA polymerase; and-   9038-9397: GBV-B 3′ non-translated region.

FIGS. 3A-3C illustrates the amino acid sequence (SEQ. ID. NO. 3) for afull-length GBV-B replicon sequence infectious in tamarins (Sbardellatiet al., J. Gen. Virol. 82:2437-2448, 2001). The approximate location ofthe GBV-B regions are provided as follows:

-   1-158 (putative): structural protein core, nucleocapsid protein;-   159 (putative)-348 (putative): structural protein E1, envelope    protein;-   349 (putative)-732 (putative): structural protein E2, envelope    protein;-   733 (putative)-940: Non-structural protein 2 (NS2);-   941-1560 (putative): Non-structural protein 3 (NS3), GBV-B NS3    protease/helicase;-   1561 (putative)-1615: Non-structural protein 4A (NS4A), NS3 protease    cofactor;-   1616-1863 (putative): Non-structural protein 4B (NS4B);-   1864 (putative)-2274: Non-structural protein 5A (NSSA); and-   2275-2864: Non-structural protein 5B (NSSB); GBV-B RNA-dependent RNA    polymerase.

FIG. 4 provides a schematic representation of the GBV-B neo-RepA,neo-RepB, neo-RepC and neo-RepD constructs. The nucleotide sequencesbelow the drawing correspond to the 3′end of the GBV-B 5′UTR, thepartial core coding sequence, the nucleotides added to create arestriction site and to put the subsequent neomycin phosphotransferasegene sequences in the same translation frame of partial core sequence,and the 5′-end of neomycin phosphotransferase gene sequences. The abovedescribed sequences corresponding to neo-RepA, neo-RepB, neo-RepC andneo-RepD constructs respectively are shown as SEQ. ID. NOs. 4, 5, 6 and7. The portion of the sequence belonging to GBV-B 5′-UTR is underlined,that representing translated GBV-B sequences is bold-faced, the sequencecorresponding to added PmeI restriction site is in italic and thatcorresponding to a portion of the neomycin phosphotransferase gene is inregular characters. The translated sequences are organized in nucleotidetriplets and the corresponding amino acids are indicated below eachcognate nucleotide sequence (SEQ. ID. NOs. 8, 9, 10 and 11).

FIG. 5 illustrates the effects of human α-IFN on the replication ofGBV-B and HCV replicons. Clones designated 10A for a HCV replicon in aHuh7 cell and B76.1/Huh7 for a GBV-B replicon in a Huh7 cell werecompared. Quantitative PCR was employed using a primer set recognizingthe neomycin phosphotransferase gene. Each curve was derived normalizingthe replicon RNA amounts to those of endogenous reference GAPDH.

FIG. 6 provides an alignment of deduced amino acid sequences of HCV(SEQ. ID. NO. 12) and GBV-B (SEQ. ID. NO. 13) replicons spanning HCVadaptive mutations. Part of NS3, NS5A and NS5B protein sequences areshown. The position of HCV mutation is underlined in the wild typesequence and the mutated amino acids described in those positions(Bartenschlager et al., Antiviral Res. 52:1-17, 2001) are indicatedabove the wild type sequence. The line above the HCV NS5A sequenceindicates the amino acids missing in a deletion mutant (Blight et al.,Science. 290:1972-1974, 2000). Numbering of HCV amino acids is asdescribed in Bartenschlager et al., Antiviral Res. 52:1-17, 2001. TheR2884G mutation in NS5B is boldface.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features GBV-B replicons and replicon enhancedcells. GBV-B replicons and replicon enhanced cells have a variety ofuses including: providing tools for studying GBV-B replication,polyprotein production and polyprotein processing; identifying compoundsinhibiting GBV-B; providing a surrogate model for identifying compoundsinhibiting HCV; and providing a scaffold for producing GBV-B/HCVchimeric replicons.

Compounds inhibiting GBV-B or HCV have research and therapeuticapplications. Research applications include using viral inhibitors tostudy viral proteins, polyprotein processing or viral replication.Therapeutic applications include using those compounds havingappropriate pharmacological properties such as efficacy and lack ofunacceptable toxicity to treat or inhibit HCV infection in a patient.

The similarities between GBV-B and HCV allow for GBV-B to be used as asurrogate model for testing anti-HCV agents. An advantage of using GBV-Bas a surrogate model is its ability to infect animals such as tamarinsand owl monkeys. The generally accepted animal model for testing HCVcompounds are chimpanzees. Animals susceptible to GBV-B infection suchas tamarins and owl monkeys provide a smaller and generally more readilyavailable and less expensive model than chimpanzees.

Using GBV-B replicons and replicon enhanced cells provides an in vitromodel that can be used to screen for antiviral compounds prior toinfecting an animal susceptible to GBV-B. The animal can then beinfected with a GBV-B virus.

GBV-B Replicons

GBV-B replicons are RNA molecules able to autonomously replicate in acultured cell and produce detectable levels of one or more GBV-Bproteins. GBV-B replicons contain RNA molecules coding for thefull-length GBV-B genome or a subgenomic construct. In an embodiment ofthe present invention the replicon can replicate in a human hepatomacell, preferably, Huh7 or Hep3B.

A GBV-B replicon may contain non-GBV-B sequences in addition to GBV-Bsequences. The additional sequences should not prevent replication andexpression, and preferably serve a useful function. Sequences that canbe used to serve a useful function include a selection sequence, areporter sequence, transcription elements and translation elements.

GBV-B Genomic and Subgenomic Regions

GBV-B genomic and subgenomic constructions contain a GBV-B 5′UTR, aGBV-B NS3-NS5B polyprotein encoding region and a GBV-B 3′ UTR. GBV-Bgenomic constructs also contain a region coding for the structural GBV-Bproteins and NS2, while GBV-B subgenomic constructs may also contain allor a portion of the structural region starting at the N-terminal coreregion and/or NS2. Preferably, the GBV-B genomic construct can producetamarin infectious GBV-B virions in culture.

The NS3-NS5B polyprotein encoding region provides for a polyprotein thatcan be processed in a cell into different proteins. Suitable NS3-NS5Bpolyprotein sequences that may be part of a replicon include thosepresent in different GBV-B strains and functional equivalents thereofresulting in the processing of NS3-NS5B to a produce functionalreplication machinery. Proper processing can be measured by assaying,for example, NS5B RNA-dependent RNA polymerase, NS3 protease activity orNS3 helicase activity.

The NS3-NS5B polyprotein region also includes either an initial Mettranslation initiation codon (AUG) or an upstream region able to betranslated. An example of such an upstream region is a NS2 regioncontaining a Met translation initiation codon.

The GBV-B 5′ UTR region provides an internal ribosome entry site forprotein translation and elements needed for replication. In subgenomicreplicons, a partial GBV-B core sequence of at least about 21nucleotides from the start of the core sequence appears to increasereplicon transfection efficiency into cells that are not repliconenhanced. An increase in replicon activity was found to correlate withthe length of the partial core sequence inserted before a reporter orselector gene. In different embodiments the partial core sequence is 3′of an AUG codon and is at least about 21 nucleotides, at least about 39nucleotides, at least about 63 nucleotides, or about 21-63 nucleotidesare present.

In multi-cistronic replicons two or more internal ribosome entry siteelements can be present. The internal ribosome entry site towards the 5′end of the replicon should be a GBV-B 5′ UTR. Additional internalribosome entry site elements that are present can be non-GBV-B internalribosome entry site elements. Examples of non-GBV-B internal ribosomeentry site elements that can be used are the EMCV internal ribosomeentry site, poliovirus internal ribosome entry site, and bovine viraldiarrhea virus internal ribosome entry site.

The GBV-B 3′ UTR assists GBV-B replication. GBV-B 3′ UTR includesnaturally occurring GBV-B 3′ UTR and functional derivatives thereof. Thefull length GBV-B 3′ UTR is described by Traboni, InternationalPublication Number WO/73466, International Publication Date 7 Dec. 2000,and Bukh et al., International Publication Number WO/75337,International Publication Date 14 Dec. 2000.

Preferred GBV-B replicons contain a GBV-B sequence able to infect newworld monkeys, preferably tamarins and/or owl monkeys. Examples ofinfectious GBV-B sequences include those provided by Bukh et al.,Virology 262:470478, 1999 and Bukh et al., International PublicationNumber WO/75337, International Publication Date 14 Dec. 2000; and bySbardellati et al., J. Gen. Virol. 82:2437-2448, 2001, and Traboni,International Publication Number WO/73466, International PublicationDate 7 Dec. 2000.

Modifications to an infectious GBV-B replicon sequence can be createdusing standard techniques to produce, for example, additional infectiousGBV-B replicons. Modifications for additional infectious GBV-B repliconsprovide for one or more nucleic acid substitution(s), insertion(s),deletion(s) or a combination thereof. Different modifications can bedesigned taking into account nucleic acid sequences and encoded aminoacid sequences of different GBV-B sequences; variable and conservedGBV-B amino acid and nucleic acids; and can be experimentally created.

Experimentation to obtain a functional replicon sequence can beperformed by introducing a functional replicon into a cell and isolatinga replicon from a transfected cell. The spontaneous mutation rate of thereplicon RNA sequence will provide different mutations. Those mutationscompatible with a functional replicon are selected for by obtaining areplicon expressing cell. The sequence of mutations can be identifiedand used to produce additional functional replicons.

In different embodiments of the present invention the GBV-B repliconencodes a GBV-B NS3-NS4A-NS4B-NS5A-NS5B sequence substantially similarto residues 941-2864 of SEQ. ID. NO. 3 or a NS2-NS3-NS4A-NS4B-NS5A-NS5Bsequence substantially similar to residues 733-2864 of SEQ. ID. NO. 3.Substantially similar amino acid sequences have a sequence identity ofat least 85%, at least 95%, at least 99%, or 100%; and/or differ fromeach other by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 amino acids.

Amino acid differences between polypeptides can be calculated bydetermining the minimum number of amino acid modifications in which thetwo sequences differ. Amino acid modifications can be deletions,additions, substitutions or any combination thereof.

Amino acid sequence identity can be determined by methods well known inthe art that compare the amino acid sequence of one polypeptide to theamino acid sequence of a second polypeptide and generate a sequencealignment. Amino acid identity can be calculated from the alignment bycounting the number of aligned residue pairs that have identical aminoacids.

Methods for determining sequence identity include those described bySchuler, G. D. in Bioinformatics: A Practical Guide to the Analysis ofGenes and Proteins, Baxevanis, A. D. and Ouelette, B. F. F., eds., JohnWiley & Sons, Inc, 2001; Yona et al., in Bioiiformatics: Sequence,structure and databanks, Higgins, D. and Taylor, W. eds., OxfordUniversity Press, 2000; and Bioinformatics: Sequence and GenomeAnalysis, Mount, D. W., ed., Cold Spring Harbor Laboratory Press, 2001.Methods to determine amino acid sequence identity are codified inpublicly available computer programs such as GAP (Wisconsin PackageVersion 10.2, Genetics Computer Group (GCG), Madison, Wisc.), BLAST(Altschul et al., J. Mol. Biol. 215(3):403-10, 1990), and FASTA(Pearson, Methods in Enzymology 183:63-98, 1990, R. F. Doolittle, ed.).

In an embodiment of the present invention sequence identity between twopolypeptides is determined using the GAP program (Wisconsin PackageVersion 10.2, Genetics Computer Group (GCG), Madison, Wisc.). GAP usesthe alignment method of Needleman and Wunsch. (Needleman et al., J. Mol.Biol. 48:443453, 1970.) GAP considers all possible alignments and gappositions between two sequences and creates a global alignment thatmaximizes the number of matched residues and minimizes the number andsize of gaps. A scoring matrix is used to assign values for symbolmatches. In addition, a gap creation penalty and a gap extension penaltyare required to limit the insertion of gaps into the alignment. Defaultprogram parameters for polypeptide comparisons using GAP are theBLOSUM62 (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919,1992) amino acid scoring matrix (MATrix=blosum62.cmp), a gap creationparameter (GAPweight=8) and a gap extension pararameter(LENgthweight=2).

Multi-Cistronic Configurations

Multi-cistronic replicons can be produced having differentconfigurations. The different configurations can vary, for example, inthe placement of a selection or reporter gene, the placement ofnon-structural genes, the placement and presence of structural regions,and the presence of more than two cistrons.

In an embodiment of the present invention, the GBV-B replicon is capableof replication in a cell, such as a human hepatoma cell, preferably aHuh7 cell; and comprises the following regions:

-   -   a GBV-B 5′ UTR substantially similar to bases 1445 of SEQ. ID.        NO. 1;    -   a selection or reporter sequence functionally coupled to the        GBV-B 5′ UTR;    -   an internal ribosome entry site;    -   a NS3-NS5B sequence substantially similar to bases 1938-7709 of        SEQ ID NO: 1 functionally coupled to the internal ribosome entry        site and a AUG translation initiation codon; and    -   a GBV-B 3′ UTR substantially similar to bases 7710-8069 of SEQ.        ID. NO. 1.

Additional embodiments concerning the GBV-B replicon include one or moreof following:

-   -   (1) The presence of a GBV-B structural region contiguous with        the GBV-B 5′ UTR is present. The structural region may contain        an additional region, such as NS2. Preferably, the GBV-B        structural region comprises a sequence substantially similar to        a sequence selected from the group consisting of: bases 446-511        of SEQ. ID. NO. 1, bases 446-487 of SEQ. ID. NO. 1, bases of        446-469 of SEQ. ID. NO. 1, the RNA version of bases 446-2641        (core-E2/p7) of SEQ. ID. NO. 2 and the RNA version of bases        446-3265 (core-NS2) of SEQ. ID. NO. 2;    -   (2) The presence of a GBV-B NS2 region or core region contiguous        with the 5′ end of the NS3-NS5B region, preferably the NS2        region if present has a sequence substantially similar to the        RNA version of bases 2642-3265 of SEQ. ID. NO. 2;    -   (3) The GBV-B 3′ UTR has a sequence substantially similar to        bases 7710-8069 of SEQ. ID. NO. 1;    -   (4) The internal ribosome entry site has a sequence        substantially similar to bases 1324-1934 of SEQ. ID. NO. 1; and    -   (5) The GBV-B replicon consists of the GBV-B 5′ UTR, a GBV-B        structural region (which may include NS2), the selection or        reporter sequence, the internal ribosome entry site, an NS3-NS5B        or NS2-NS5B sequence, and a GBV-B 3′ UTR.

Reference to “the RNA version” indicates a ribose backbone and thepresence of uracil instead of thymine.

Reference to a GBV-B region is not limited to a naturally occurringGBV-B region, but also includes derivatives of such regions. The scopeof the derivatives is provided by a relationship (substantially similar)to a reference sequence.

Substantially similar nucleotide sequences have a nucleotide sequenceidentity of at least 85%, at least 95%, at least 99%, or 100%; and/ordiffer from each other by 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-25, 1-30,1-35, 140, 1-45, or 1-50 nucleotides. Nucleotide differences between twosequences can be calculated by determining the minimum number ofnucleotide modifications in which the two sequences differ. Nucleotidemodifications can be deletions, additions, substitutions or anycombination thereof. A preferred additional nucleotide sequence is a 5′AUG sequence next to a coding sequence lacking a 5′ AUG.

Nucleotide sequence identity can be determined by methods well known inthe art that compare the nucleotide sequence of one sequence to thenucleotide sequence of a second sequence and generate a sequencealignment. Sequence identity can be determined from the alignment bycounting the number of aligned positions having identical nucleotides.

Methods for determining nucleotide sequence identity between twopolynucleotides include those described by Schuler, in Bioinformatics: APractical Guide to the Analysis of Genes and Proteins, Baxevanis, A. D.and Ouelette, B. F. F., eds., John Wiley & Sons, Inc, 2001; Yona et al.,in Bioinformatics: Sequence, structure and databanks, Higgins, D. andTaylor, W. eds., Oxford University Press, 2000; and Bioinformatics:Sequence and Genome Analysis, Mount, D. W., ed., Cold Spring HarborLaboratory Press, 2001. Methods to determine nucleotide sequenceidentity are codified in publicly available computer programs such asGAP (Wisconsin Package Version 10.2, Genetics Computer Group (GCG),Madison, Wisc.), BLAST (Altschul et al., J. Mol. Biol. 215(3):403-10,1990), and FASTA (Pearson, W. R., Methods in Enzymology 183:63-98, 1990,R. F. Doolittle, ed.).

In an embodiment of the present invention, sequence identity between twopolynucleotides is determined by application of GAP (Wisconsin PackageVersion 10.2, Genetics Computer Group (GCG), Madison, Wisc.). GAP usesthe alignment method of Needleman and Wunsch. (Needleman et al., J. Mol.Biol. 48:443-453, 1970.) GAP considers all possible alignments and gappositions between two sequences and creates a global alignment thatmaximizes the number of matched residues and minimizes the number andsize of gaps. A scoring matrix is used to assign values for symbolmatches. In addition, a gap creation penalty and a gap extension penaltyare required to limit the insertion of gaps into the alignment. Defaultprogram parameters for polynucleotide comparisons using GAP are thenwsgapdna.cmp scoring matrix (MATrix=nwsgapdna.cmp), a gap creationparameter (GAPweight-50) and a gap extension pararameter(LENgthweight=3).

Selection Sequence

A selection sequence in a GBV-B replicon can be used to facilitate theproduction of GBV-B replicon enhanced cells and replicon maintenance ina cell. Selection sequences are typically used in conjunction with someselective pressure that inhibits growth of cells not containing theselection sequence. Examples of selection sequences include sequencesencoding antibiotic resistance and ribozymes.

Antibiotic resistance can be used in conjunction with an antibiotic toselect for cells containing replicons. Examples of selection sequencesproviding for antibiotic resistance are sequences encoding resistance toneomycin, hygromycin, puromycin, or zeocin.

A ribozyme serving as a selection sequence can be used in conjunctionwith an inhibitory nucleic acid molecule that prevents cellular growth.The ribozyme recognizes and cleaves the inhibitory nucleic acid.

Reporter Sequence

A reporter sequence can be used to detect replicon replication orprotein expression. Preferred reporter proteins are enzymatic proteinswhose presence can be detected by measuring product produced by theprotein. Examples of reporter proteins include, luciferase,beta-lactamase, secretory alkaline phosphatase, beta-glucuronidase, andgreen fluorescent protein and its derivatives. In addition, a reporternucleic acid sequence can be used to provide a reference sequence thatcan be targeted by a complementary nucleic acid probe. Hybridization ofthe probe to its target can be determined using standard techniques.

Additional Sequence Configuration

Additional sequences are preferable 5′ or 3′ of a GBV-B genome orsubgenomic genome region. However, the additional sequences can belocated within a GBV-B genome as long as the sequences do not preventdetectable replicon activity. If desired, additional sequences can beseparated from the replicon by using a ribozyme recognition sequence inconjunction with a ribozyme.

Additional sequences can be part of the same cistron as the GBV-Bpolyprotein or can be a separate cistron. If part of the same cistron,the additional sequences coding for a protein should result in a productthat is either active as a chimeric protein or is cleaved inside a cellso it is separated from a GBV-B protein.

Selection and reporter sequences encoding a protein when present as aseparate cistron should be associated with elements needed fortranslation. Such elements include a 5′ ribosome entry site.

Replicon Encoding Nucleotide Sequence

GBV-B replicons can be produced from a nucleic acid molecule coding forthe replicon. A nucleic acid molecule can be single-stranded or part ofa double strand, and can be RNA or DNA. Depending upon the structure ofthe nucleic acid molecule, the molecule may be used as a replicon or inthe production of a replicon. For example, single-stranded RNA havingthe proper regions can be a replicon, while double-stranded DNA thatincludes the complement of a sequence coding for a replicon or repliconintermediate may useful in the production of the replicon or repliconintermediate.

Nucleic acid containing a sequence coding for a replicon can be producedfrom an expression vector. Replicons can be introduced into a cell as anRNA molecule in vitro transcribed from a corresponding DNA cloned in anexpression vector, or can be isolated from a first cell expressing theexpression vector and then transfected into a second cell.

An expression vector contains recombinant nucleic acid encoding adesired sequence along with regulatory elements for proper transcriptionand processing. The regulatory elements that may be present includethose naturally associated with the nucleotide sequence encoding thedesired sequence and exogenous regulatory elements not naturallyassociated with the nucleotide sequence. Examples of expression vectorsare cloning vectors, modified cloning vectors, specifically designedplasmids and viruses.

Starting with a particular amino acid sequence and the known degeneracyof the genetic code, a large number of different encoding nucleic acidsequences can be obtained. The degeneracy of the genetic code arisesbecause almost all amino acids are encoded by different combinations ofnucleotide triplets or “codons”. The translation of a particular codoninto a particular amino acid is well known in the art (see, e.g., LewinGENES IV, p. 119, Oxford University Press, 1990). Amino acids areencoded by codons as follows:

-   A=Ala-Alanine: codons GCA, GCC, GCG, GCU-   C=Cys=Cysteine: codons UGC, UGU-   D=Asp=Aspartic acid: codons GAC, GAU-   E=Glu=Glutamic acid: codons GAA, GAG-   F-Phe-Phenylalanine: codons UUC, UUU-   G=Gly=Glycine: codons GGA, GGC, GGG, GGU-   H=His=Histidine: codons CAC, CAU-   I=Ile=Isoleucine: codons AUA, AUC, AUU-   K=Lys=Lysine: codons AAA, AAG-   L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU-   M=Met=Methionine: codon AUG-   N=Asn=Asparagine: codons AAC, AAU-   P=Pro=Proline: codons CCA, CCC, CCG, CCU-   Q=Gln=Glutamine: codons CAA, CAG-   R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU-   S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU-   T=Thr-Threonine: codons ACA, ACC, ACG, ACU-   V=Val=Valine: codons GUA, GUC, GUG, GUU-   W=Trp=Tryptophan: codon UGG-   Y=Tyr_Tyrosine: codons UAC, UAU.

Detection Methods

Methods for detecting replicon activity include those measuring theproduction or activity of replicon RNA and encoded protein. Measuringincludes qualitative and quantitative analysis.

Techniques suitable for measuring RNA production include those detectingthe presence or activity of RNA. The presence of RNA can be detectedusing, for example, complementary hybridization probes or quantitativePCR. Techniques for measuring hybridization between complementarynucleic acid and quantitative PCR are well known in the art. (See forexample, Ausubel, Current Protocols in Molecular Biology, John Wiley,1987-1998, Sambrook et al., Molecular Cloning, A Laboratory Manual,2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989, and U.S. Pat.No. 5,731,148.)

RNA enzymatic activity can be provided to the replicon by using aribozyme sequence. Ribozyme activity can be measured using techniquesdetecting the ability of the ribozyme to cleave a target sequence.

Techniques for measuring protein production include those detecting thepresence or activity of a produced protein. The presence of a particularprotein can be determined by, for example, immunological techniques.Protein activity can be measured based on the activity of a GBV-Bprotein or a reporter protein sequence.

Techniques for measuring GBV-B protein activity vary depending upon theprotein that is measured. Techniques for measuring the activity ofdifferent non-structural proteins such as NS3 and NSSB, are well knownin the art. (See, for example, the references provided in the Backgroundof the Invention.) Assays measuring replicon activity also include thosedetecting virion production from a replicon that produces a virion; andthose detecting a cytopathic effect from a replicon producing proteinsexerting such an effect. Cytopathic effects can be detected by assayssuitable to measure cell viability.

Assays measuring replicon activity can be used to evaluate the abilityof a compound to modulate GBV-B activities. Such assays can be carriedout by providing one or more test compounds to a cell expressing a GBV-Breplicon and measuring the effect of the compound on replicon activity.If a preparation containing more than one compound is found to modulatereplicon activity, individual compounds or smaller groups of compoundscan be tested to identify replicon active compounds.

Compounds identified as inhibiting GBV-B activity can be used to producereplicon enhanced cells and may be therapeutic or research compounds.The ability of a compound to serve as a therapeutic compound for HCV canbe confirmed using animals susceptible to GBV-B and, if desired, throughthe subsequent use of a chimpanzee infected with HCV.

Replicon Enhanced Cells

Replicon enhanced cells have an increased ability to maintain areplicon. Replicon enhanced cells can be produced by selecting for acell able to maintain a GBV-B replicon and then curing the cell of thereplicon.

Initial transfection can be performed using a replicon having awild-type GBV-B sequence that contains at least a NS3-NS5B sequence or afunctional derivative thereof. The replicon preferably contains aselection sequence to facilitate replicon maintenance.

Cells can be cured of replicons using different techniques such as thoseemploying a replicon inhibitory agent. Replicon inhibitory agentsinhibit replicon activity or select against a cell containing areplicon. Examples of such agents include IFN-α and compounds found toinhibit GBV-B replicon activity. The ability of a cured cell to be areplicon enhanced cell can be measured by introducing a replicon intothe cell and determining efficiency of replicon maintenance andactivity.

A first GBV-B replicon introduced into a replicon cured GBV-B cell maybe the same or different than a second GBV-B replicon introduced intothe GBV-B replicon enhanced cell. The two replicons can differ by theGBV-B coding sequences or by other sequences that may be present such asa selection sequence or a reporter sequence. Preferably, the first GBV-Breplicon introduced into a GBV-B replicon enhanced cell has the sameGBV-B sequences as the replicon that was used to produce the enhancedcell.

The overall method for producing a replicon enhanced cell can besummarized as involving the steps of: (a) introducing and maintaining aGBV-B replicon into a cell and (b) curing the cell of the replicon. Inan embodiment of the present invention, the method further comprises:step (c) introducing and maintaining a GBV-B replicon into a cell,wherein the replicon may be the same or different from the step (a)replicon; and step (d) curing the cell of the replicon used in step (b),wherein the curing may be performed the same way or different from thetechnique employed in step (b). In a preferred embodiment for theproduction of an enhanced cell supporting a genomic replicon, step (a)is performed using a subgenomic replicon and step (c) is preformed usinga genomic replicon.

Virion Production

Genomic replicons can be used to produce virions. The produced virionshave different uses such as providing for activities that can bemeasured and as a source of virus for infecting animals. Measuringvirion activities under different conditions can be used to gain abetter understanding of virion production and to assay the ability of acompound to alter such activities.

Preferably, genomic replicons are used to produce virions in repliconenhanced cells. Genomic replicons that can be used for virion productioninclude those having a single cistron and those having multiplecistrons.

GBV-B/HCV Chimerics

Chimeric GBV-B/HCV replicons infectious in a GBV-B susceptible animalprovide for a further enhancement in using the animal as a surrogatemodel. The chimeric GBV-B/HCV replicon sequence would contain one ormore HCV protein encoding regions or a portion thereof in a GBV-Bscaffold. The GBV-B and HCV regions corresponding to 5′ UTR, core, E1,E2, NS2, NS3, NS4A, NS4B, NSSA, NS5B, and 3′UTR regions are well knownin the art. (See, for example, references cited in the Background of theInvention and Hong et al., U.S. Publication Number U.S. 2001/0034019,Publication Date Oct. 25, 2001).

One or more GBV-B regions or a portion thereof present in a replicondescribed herein can be replaced with the corresponding region from HCV.In different embodiments the corresponding region encodes at least about50 amino acids, at least about 75 amino acids, or an entire regionpresent in a naturally occurring HCV.

Numerous examples of naturally occurring HCV isolates are well known inthe art. HCV isolates can be classified into the following six majorgenotypes comprising one or more subtypes: HCV-1/(1a, 1b, 1c),HCV-2/(2a, 2b, 2c), HCV-3/(3a, 3b, 10a), HCV-4/(4a), HCV-5/(5a) andHCV-6/(6a, 6b, 7b, 8b, 9a, 11a). (Simmonds, J. Gen. Virol., 693-712,2001.) Examples of particular HCV sequences such as HCV-BK, HCV-J,HCV-N, HCV-H, have been deposited in GenBank and described in variouspublications. (See, for example, Chamberlain et al., J. Gen. Virol.,1341-1347, 1997).

Replicon enhanced cells can be used to screen for chimeric GBV-B/HCVreplicon sequences that can replicate and process viral polyprotein in acell. The ability of functional chimeric GBV-B/HCV replicons to infectan animal such as a tamarin or owl monkey can then be evaluated.

EXAMPLES

Examples are provided below to further illustrate different features ofthe present invention. The examples also illustrate useful methodologyfor practicing the invention. These examples do not limit the claimedinvention.

Example 1 Techniques

This example illustrates techniques that can be employed for producingand analyzing GBV-B replicons and replicon enhanced cells.

Cell Lines and Culture Conditions

The human hepatoma cell line Huh7 was grown in high glucose Dulbecco'smodified Eagle medium (DMEM; Life Technologies) supplemented with 2 mML-glutamine, 100 U/ml of penicillin, 100 μg/ml streptomycin, 10% fetalbovine serum. Cells were subcultivated twice a week with a 1:5 splitratio. Aoutus trivirgatus (owl monkey) kidney cell line (OMK 637-69;ATCC number CRL-1556) was grown in minimum essential medium in Earle'sBSS with non essential amino acids (MEM; Life Technologies) supplementedwith 100 U/ml of penicillin, 100 μg/ml streptomycin, 10% fetal bovineserum. Saguinus oedipus (tamarin) lymphoblast cell line B95-8a, kindlyprovided by Dr. Fumio Kobune, was grown in RPMI 1640 medium (RPMI; LifeTechnologies) supplemented with 2 mM L-glutamine, 100 U/ml ofpenicillin, 100 μg/ml streptomycin, 10 mM HEPES, 1.0 mM sodium pyruvate,10% fetal bovine serum. Neomycin-resistant lines were grown in thepresence of G418 final concentration ranging between 0.250 and 1 mg/ml.

Plasmids Construction

GBV-B subgenomic replicon constructs were obtained by replacing theregions coding for structural proteins and NS2 protein with thesequences of neomycin phosphotransferase gene (neo) and EMCV internalribosome entry site in the plasmid FL3/pACYC177 (EMBL accession numberAJ277947). The FL3/pACYC177 plasmid encodes a GBV-B infectiousfull-length cDNA downstream of a T7 polymerase promoter. Neo and theEMCV internal ribosome entry site were joined stepwise to the GBV-Bsequences by assembly-PCR and ligation reactions creating a unique AscIsite at the junction between GBV-B 5′UTR and neo-gene.

The final GBV-B replicon sequence was moved as a BamHI-XhoI fragmentinto the more versatile pGBT9 vector (Clontech). Two SapI sites wereremoved from the neo-gene by primer-based mutagenesis leaving a SapIsite at the 3′-end of the GBV-B coding sequence, useful for run-offtranscription.

Four constructs, GBV-B-neo-RepA (neo-RepA), GBV-B-neo-RepB (neo-RepB),GBV-B-neo-RepC (neo-RepC) and GBV-B-neo-RepD (neo-RepD) were produced.Neo-Rep A contains the GBV-B 5′UTR followed by neo-gene. Neo-RepB,neo-repC and neo-repD contain the GBV-B 5′UTR, the ATG start codon andthe subsequent 21, 39 and 63 nucleotides respectively of the GBV-B corecoding sequence upstream of neo gene. The N-terminus of the neomycinphosphotransferase protein resulting from the described cloning designis preceded by three amino acids, depending on the addition of a cloningsite upstream of neomycin phosphotransferase gene. Neo-RepB, neo-RepCand neo-RepD were obtained by replacing the original BamHI-AscI fragmentof the neo-RepA clone with a BamHI-AscI fragment containing the ATGstart codon and subsequent 21, 39 and 63 nucleotides of the GBV-B corecoding sequence respectively. The RepD sequence is provided in FIG. 1.

Chimeric replicons bearing the HCV NS5B gene in place of the GBV-Bcorresponding gene were constructed in pGBT9 vector. Construction wasachieved by replacing the SfiI-XhoI fragment of the GBV-B RepB clone,spanning NS5B region, with the corresponding fragment from a full-lengthchimeric clone containing NS5B of HCV, genotype 1a.

Bla-RepA, bla-RepB, bla-RepC, bla-RepD constructs were producedcontaining the β-lactamase gene (bla) in place of neo. The bla-Repreplicons was constructed by replacing in the GBV-B neo-Rep constructsthe AscI-PmeI fragment spanning neo-gene with an AscI-PmeI fragmentincluding the β-lactamase gene.

Mutants in the polymerase active site GDD motif were obtained by firstconstructing a GDD to GAA mutated neo-RepB clone by means ofprimer-based mutagenesis and subsequently replacing a restrictionfragment spanning the mutation into the other wild type constructs.

RT-PCR amplification products of RNA from RepB76.1/Huh7 cells weresubcloned in the pCR2.1 vector to perform sequencing.

GBV-B genomic replicon constructs neo-FL-A, neo-FL-B, neo-FL-C andneo-FL-D (see FIG. 4 for neo-FL-D partial sequence, corresponding toneo-RepD partial sequence) were obtained by inserting the sequences ofneomycin phosphotransferase gene (neo) and EMCV internal ribosome entrysite within the plasmid FL3/pACYC177 (EMBL accession number AJ277947)upstream of the regions coding for the GBV-B structural proteins bymeans of routine molecular biology techniques. Neo-FL-A contains theGBV-B 5′UTR followed by neo-gene. Neo-FL-B, neo-FL-C and neo-FL-Dcontain the GBV-B 5′UTR, the ATG start codon and the subsequent 21, 39and 63 nucleotides respectively of the GBV-B core coding sequenceupstream of neo gene, as well as the corresponding neo-Rep subgenomicconstructs. The N-terminus of the neomycin phosphotransferase proteinresulting from the described cloning design is preceded by three aminoacids, depending on the addition of a cloning site upstream of neomycinphosphotransferase gene.

Sequence Analysis

Sequencing was performed by the Big Dye Terminator Cycle sequencing kitwith AmplyTaq (Applied Biosystems) and run with an Applied Biosystemsmodel 373A sequencer.

In Vitro Transcription

SapI-linearized plasmids encoding G13V-B replicons were invitro-transcribed by T7 RNA polymerase using an Ambion Megascript kitunder nuclease-free conditions following the manufacturer'sinstructions. The reaction was terminated by incubation with DNAse I andprecipitation with LiCl, according to the manufacturer's instructions.RNA was resuspended in nuclease free water, quantified by absorbance at260 nm, immediately frozen in dry ice in 10 μg aliquots and stored at−80° C.

Electroporation Method and Monitoring of Replication

Human hepatoma Huh7 and derived cell lines, as well as monkey cell lineswere used to test replication of GBV-B molecular constructs. Confluentcells from 15 cm diameter plate were divided 1:2. Cells were recoveredafter 24 hours in 5 ml medium, washed twice with 40 ml cold DEPC-treatedPBS, filtered with Cell Striner filters (Falcon) and diluted in coldDEPC-treated PBS at a concentration of 10⁷ cells/ml. 2×10⁶ cell aliquotswere subjected to electroporation with 10 μg of in vitro transcribed RNAby 2 pulses at 0.35 KV and 10 μF using a BioRad Genepulser II.

Immediately after electric pulses cells were diluted in 8 ml completeDulbecco's Modified Eagle Medium (DMEM) and processed with differentprotocols depending on the selection/tracer used. In the case ofneo-constructs transformation, cells were divided in 3 plates of 15 cmdiameter and on the following day the selecting antibiotic G418 (SigmaG-9516) was added at a concentration of 0.250, 0.5 or 1 mg/ml. In 2weeks neomycin-sensitive cells died and at the fourth week survivingcell clones were observed for the 0.25 mg/ml concentration. Survivingclones were picked-up and expanded by growing them in individual plates.When a bla reporter gene was used, 1.5 ml, 1.0 ml and 0.5 ml oftransfected cells suspension were plated in each well ofMultiwell-6-wells plate (Falcon cat. N. 35-3046) to be stainedrespectively at 24, 48 and 72 hours.

Aurora substrate system “CCF4” was used to measure β-lactamase activity.When quantitative PCR was used to measure transient replication, cellswere plated 1-2×10⁵/well in “6-multi-well plate”. After 3 days total RNAwas purified as described in the TRIzol protocol (Life Technologies) and10 out of 100 μl of total RNA were used in individual TaqMan reactions.

TaqMan Quantification of GBV-B RNA

GBV-B RNA was quantified by a real-time 5′ exonuclease PCR (TaqMan)assay using a primer/probe set that recognized a portion of the GBV-B5′UTR. The primers (GBV-B-F3, GTAGGCGGCGGGACTCAT (SEQ. ID. NO. 14), andGBV-B-R3, TCAGGGCCATCCAAGTCAA (SEQ. ID NO. 15)) and probe(GBV-B-P3,6-carboxyfluorescein-TCGCGTGATGACAAGCGCCAAG (SEQ. ID. NO.16)-N,N,N′,N′-tetramethyl-6-carboxyrhodamine) were selected using thePrimer Express software (PE Applied Biosystems). The fluorescent probewas obtained from PE Applied Biosystems. The primers were used at 10pmol/50 μl reaction, and the probe was used at 5 pmol/50 μl reaction.

PCR was were performed using a TaqMan Gold RT-PCR kit (PE AppliedBiosystems). PCR included a 30 minute reverse transcription step at 48°C., followed by 10 minutes at 95° C. and by 40 cycles of amplificationusing the universal TaqMan standardized conditions (a 15 second at 95°C. denaturation step followed by a 1 minute 60° C. annealing/extensionstep).

RNA transcribed from a plasmid containing the first 2000 nucleotides ofthe GBV-B genome was used as a standard to establish genome equivalents.Standard RNA was transcribed using a 17 Megascript kit (Ambion) and waspurified by DNase treatment, phenol-chloroform extraction, Sephadex-G50filtration and ethanol precipitation. RNA was quantified by absorbanceat 260 nm and stored at −80° C.

All reactions were run in duplicate by using the ABI Prism 7700 SequenceDetection System (PE Applied Biosystems). A primer set for human GAPDHmRNA (PE Applied Biosystems) was used as an endogenous reference.Transfected RNA obtained by in vitro transcription of mutant constructsin which the sequence coding for the GDD motif in the active site ofNS5B polymerase was replaced by GAA was used as calibrator. Results fromtwo independent experiments were analyzed using both the Comparative CtMethod and the standard curve method.

Preparation of Proteins, Genoinic DNA and Total RNA

Total RNA, genomic DNA and total proteins were purified from cells grownin monolayer with TRIzol reagent (Life Technologies) following themanufacturer's instructions.

Northern Blot

8 μg of total RNA extracted from Huh7 cells and Huh7-derived GBV-Breplicon cell clones was subjected to electrophoresis on a 1%agarose/formaldehyde gel, blotted onto Amersham's Hybond-N⁺ membranesand hybridized to a GBV-B RNA probe. Electrophoresis, blot andhybridization procedures were performed following the protocols ofAmersham's Hybond-N⁺ membranes instruction manual with slightmodifications. The [α³²P]-CTP-labelled RNA probe was produced by invitro transcription of a GBV-B genome fragment (nt 4641-6060 of the FL3genome sequence) cloned in pCR2.1 vector under the T7 promoter in theorientation producing a negative stranded transcript.

Non-Quantitative RT-PCR

Total RNA was used for first strand cDNA synthesis by Superscript IIreverse transcriptase (Gibco-BRL) under the manufacture's conditions.PCR amplification was performed using Elongase enzyme mix (Gibco-BRL) orTaq DNA polymerase (Promega). Primers were purchased from MWG (Germany).

Test of Putative Inhibitors of GBV-B Replication

Huh7 cell clones carrying GBV-B or HCV replicons were used to test theeffect of human interferon alpha-2b. Cells (1×10⁵) were plated into eachwell of a series of wells of Multiwell-6-wells plates (falcon cat N.35-3046) in medium without G418. After 16 hours the medium was discardedand increasing concentrations of the test compound in fresh medium wereadded to each series of wells. Controls were run using the specificcompound solvent at the appropriate dilution.

Cells were grown up to 3 days in the presence of compounds or compoundsolvent without a compound, avoiding cell confluence, and finally lysedwith TRIzol. Total RNA was purified as described in the TRizol protocol(Life Technologies). Ten out of 100 μl of total RNA were used in eachreaction.

Taq Man analysis was performed using a neomycin primer set (taq NEO 1,GATGGATTGCACGCAGGTT (SEQ. ID. NO. 17), and taq NEO 5,CCCAGTCATAGCCGAATAGCC (SEQ. ID. NO. 18)) and a NEO probe(1,6-carboxyfluorescein-TCCGGCCGCTTGGGT GGAG (SEQ. ID. NO.19)-N,N,N′,N′-tetramethyl-6-carboxyrhodamine). Human GAPDH mRNAquantified with a specific primer set (PE Applied Biosystems) was usedas an endogenous reference. GBV-B RNA extracted from mock-treated cellswas used as a calibrator. Results from two independent experiments wereanalyzed using both the Comparative Ct Method and the standard curvemethod.

Western Blot

Protein extracts were prepared from 1×10⁶ cells by TRIzol extraction andfractionated on a 10% SDS-PAGE (30-100 μg/slot). The gel was blottedonto a nitrocellulose filter by routine methodologies. The filter wasincubated with a 1:50 dilution of a pool of four tamarin sera previouslytested as immunoreactive against GBV-B antigens (Sbardellati et al., J.Gen. Virol. 82:2437-2448, 2001) in blocking buffer (5% non-fat dry milk,0.05% Tween-20 in TBS). The filter was washed 5 times with blockingbuffer and was then incubated with a mouse anti-monkey antibody (Sigma).After 5 more washes the filter was incubated with a HRP-conjugated mouseantibody and finally treated with West Pico Supersignal chemiluminescentsubstrate (PIERCE) following manufacturer's instructions and the signalsdetected by X-ray film exposure.

Example 2 Cloning GBV-B Neo-Resistant Replicons and Transfection ofMammalian Cells

The FL-3 plasmid was used as a parental molecule to build-up GBV-Bsubgenomic replicon constructs. The FL-3 plasmid encodes a tamarininfectious GBV-B genomic sequence. (Sbardellati et al., J. Gen. Virol.82:2437-2448, 2001).

GBV-B bicistronic replicons were designed as schematized in FIG. 4. Inthe first cistron the GBV-B 5′UTR sequence directs translation of theneomycin phosphotransferase selectable marker; the second cistron isformed by the EMCV internal ribosome entry site directing translation ofthe GBV-B non-structural proteins from NS3 to NS5B; downstream of thecoding region is the complete GBV-B 3′UTR sequence.

Four replicon versions varying in the 5′UTR/neo-gene boundary areillustrated in FIG. 4: neo-RepA, neo-RepB, neo-RepC and neo-RepD. Inneo-RepA the GBV-B internal ribosome entry site containing 5′UTR wasinserted up to the ATG polyprotein starting codon, thus directing thetranslation of neomycin phosphotransferase with no upstream GBV-B corecoding sequence. In neo-RepB a stretch of 21 nucleotides coding for thefirst 7 amino acids of core protein following ATG was included producinga neo-gene N-terminal fusion protein. In neo-RepC a stretch of 39nucleotides coding for the first 13 amino acids of core proteinfollowing ATG was included producing a neo-gene N-terminal fusionprotein. In neo-RepD a stretch of 63 nucleotides coding for the first 21amino acids of core protein following ATG was included producing aneo-gene N-terminal fusion protein. The N-terminus of the neomycinphosphotransferase protein resulting from the described cloning designin all the neo-Rep constructs was preceded by three more amino acids(GRA) depending on the addition of the cloning site upstream of neomycinphosphotransferase gene.

Neo-Rep plasmids were in vitro transcribed after linearization at anengineered SapI sites to generate GBV-B subgenomic transcriptsterminating at the precise 3′-end of the genomic infectious molecules.In vitro transcribed RNA was transfected into Huh7 human hepatoma cellsby electroporation. RNA from neo-RepB-GAA plasmid, mutated in the activesite of the NS5B polymerase, was used as a negative control.

After 24 hours of growth in the absence of selection, neomycin (G418)was added to the medium at 0.250 mg/ml. After 30 days of culture in thepresence of G418, resistant clones were picked-up and grown asindividual cell lines.

In a typical experiment, 64 neo-resistant colonies upon transfection of2×10⁶ cells with 10 μg of neo-RepA RNA were selected, 793 colonies upontransfection of neo-RepB were selected, 780 colonies upon transfectionwith neo-RepC were selected, and 1920 colonies upon transfection withneo-RepD were selected. Parallel attempts to isolate neo-resistantclones upon selection with higher G418 concentrations failed. However,the concentration of G418 could be increased for at least some of thecell clones, once the clones were isolated and individually grown.

The neo-resistant individual cell lines showed some variability in thegrowth rate. A fast-growing clone kept at 1 mg/ml G418 was designated“B76.1/Huh7”.

B76.1/Huh7 was deposited in accordance with the Budapest Treaty at theAdvanced Biotechnology Center (ABC), Interlab Cell Line Collection,(Biotechnology Dept.), Largo Rossana Benzi, 10, 16132 Genova, Italy. Thedeposited B76.1/Huh7 was assigned deposit number PD02002 and a depositdate of 22 Jan. 2002.

Attempts to reproduce successful transfection of GBV-B subgenomicreplication using as recipients primate (tamarin and owl monkey) celllines of non-hepatic origin failed. Additionally, RNA transcribed fromthe chimeric HCVpol/GBV-B replicon bearing the HCV NS5B gene in place ofGBV-B counterpart was unable to replicate in cells tested.

Example 3 Detection and Quantification of GBV-B RNA in Transfected Huh7Cells

RNA was extracted from several individual neo-RepB/Huh7 cell clones andsubjected to non-quantitative RT-PCR using various sets of primers. PCRproducts was obtained only when a reverse transcription step wasincluded, indicating that amplification was exclusively RNA-dependentand not due to the presence of residual DNA in the RNA preparation.Moreover, the integration of replicon copies in host genomic DNA wasalso excluded by lack of amplification of both GBV-B and neo-genesequences from cell clones genomic DNA preparations.

To obtain quantitative measurements of replicon RNA molecules in theneo-resistant clones, Taqman RT-PCR using primers and a probecomplementary to GBV-B 5′UTR region was performed on individual cellclones selected upon transfection of neo-RepB RNA. The results,summarized in Table 1, confirmed the RNA-dependent amplification ofreplicon sequences and showed that the number of GBV-B genomeequivalents (G.E.)/cell was variable, ranging between 30 and 100. Theraise in G418 concentration resulted in a 3-fold increase in G.E./cell,as shown in Table 1 for clone B76.1/Huh7. TABLE 1 Comparison of neo-RepBcopy numbers in individual neo-resistant cell lines. Cell line G.E./μgcell RNA mean G.E./cell B4 3.24 × 10⁶ 32.4 B57 3.43 × 10⁶ 103.0 B59 2.68× 10⁶ 80.5 B76 1.40 × 10⁶ 35.0 B76.1* 4.85 × 10⁶ 121.0 B78 1.30 × 10⁶38.9 B86 0.58 × 10⁶ 29.3Cell lines were grown at 0.250 mg/ml G418;*cells grown at 1 mg/ml G418.The amount of total cellular RNA was measured determining absorbance at260 nm.

Example 4 Detection of GBV-B Proteins

GBV-B NS3 protein produced from replicon clones was visualized byWestern blot experiments performed with extracts of individual cellclones. A pool of GBV-B-infected tamarin sera, in which seroconversionhad already been detected (Sbardellati et al., J. Gen. Virol.82:2437-2448, 2001), was used as an immunological reagent to GBV-Bproteins. The results show a specific band at the expected molecularweight for NS3 that is not present in the mock-transfected cells. Theidentification of NS3 protein was confirmed using a purified GBV-B NS3preparation as a positive control.

The reactivity of those sera to NS5B, which has a size similar to NS3,though detectable by ELISA by coating a purified antigen (Sbardellati etal., J. Gen. Virol. 82:2437-2448, 2001), was not detectable by Westernblot. This was confirmed by the lack of signal to purified NS5B used asa positive control.

Example 5 Effect of Antiviral Compounds on GBV-B RepB/Huh7 Clones

The effect of human alpha-interferon (α-IFN) and other chemicalcompounds on the GBV-B replicon system was determined to evaluate thesusceptibility of the GBV-B replicon to HCV antiviral agents. The effectof different antiviral agents were determined using B76.1/Huh7 cellsalone or in parallel with 10A cells.

Experiments were performed using 10⁵ cells of B76.1/Huh7 and 10A platedinto individual wells of 6-multiwell plate and incubated overnight inthe absence of G418. The next day, the medium was replaced with freshmedium containing serial dilutions of test compound. Cells were grown upto 3 days in the presence of the test compound or the compound solvent,taking care that confluence was not reached to avoid a specific growthinhibition. (Pietschmann et al., J. Virol. 75:1252-1264, 2001.) RNA wasextracted and TaqMan analysis was performed using a primers-probe setspecific for the neomycin gene in order to avoid any methodologicaldifference in the measurement of the HCV and GBV-B RNA molecules.

The effects of human alpha-IFN is shown in FIG. 5. Human alpha-IFN isapproved for treating hepatitis C infection and reportedly acts on HCVreplicon. (Frese et al., J. Gen. Virol. 82:723-733, 2001, Guo et al., J.Virol. 75:8516-8523, 2001.) Human alpha-IFN has a comparable effect onGBV-B and HCV replicons with an IC50 of 0.45 U/ml for GBV-B and of 0.58for HCV.

Example 6 GBV-B Replicon Sequence Variation

GBV-B replicon RNA molecules able to replicate in Huh7 showed nosequence variation with respect to the parental full-length infectiousRNA. Portions of GBV-B replicon spanning the complete replicon wereamplified by RT-PCR of total RNA of B76.1/Huh7 cells and subcloned forsequencing. Two subclones per each region obtained from independentRT-PCRs were sequenced.

No mutation was consistently found in both individual subclones analyzedper region suggesting the absence of adaptive mutations. Sporadicmutations present only in one of the two subclones of each GBV-B regionwere observed. The sporadic mutations were attributed to PCR errors. Analternative explanation it that a mixed replicon RNA population existsin this cell line in which no mutation is present in every molecule.

The position of adaptive mutations reported for an HCV replicon(Bartenschlager et al., Antiviral Res. 52:1-17, 2001) was compared withthe corresponding amino acid residue in the sequence deduced for theGBV-B replicon. Results are reported in FIG. 6 and show the presence inGBV-B of “HCV adapted” amino acids in only one case, the HCV mutationR2884G in NS5B (Lohmann et al., J. Virol. 75:1437-1449, 2001), being aglycine residue present in GBV-B replicating RNA.

Example 7 Replicon Enhanced Cells

Replicon enhanced cells had an increased transfection and replicationefficiency for GBV-B replicons. The effect of replicon enhanced cellswas evaluated by eliminating the replicon RNA from B76.1/Huh7 cells andcomparing the cured cells cB76.1/Huh7 with parental Huh7.

A B76.1/Huh7 cell culture was cured of replicon RNA using a highconcentration of human α-IFN for a time sufficient to achieve totalinhibition of replication and complete degradation of the resident GBV-Breplicon RNA molecules. B76.1/Huh7 was maintained in culture with 100U/ml α-IFN for 15 days in the absence of neomycin. The disappearance ofthe selectable RNA replicon molecules was checked at the end of thetreatment by TaqMan analysis and confirmed by the inability of the“cured” clone to grow in the presence of the selector neomycin.

The resulting cured cell line was designated “cB76.1/Huh7”. cB76.1/Huh7was transfected with in vitro transcribed RNA from the neo-RepB plasmid,and kept under neomycin selection. More than 80% of the cells used fortransfection survived, indicating an increase of productive transfectionof thousand folds respect to wild type Huh7 cells.

cB76.1/Huh7 was deposited in accordance with the Budapest Treaty at theAdvanced Biotechnology Center (ABC), Interlab Cell Line Collection,(Biotechnology Dept.), Largo Rossana Benzi, 10, 16132 Genova, Italy. Thedeposited cB76.1/Huh7 was assigned deposit number PD02001 and a depositdate of 22 Jan. 2002.

The increased efficiency of replication of RepB RNA in cB76.1/Huh7 cellscompared to wild type Huh7 cells was also monitored using a colorimetricassay after transfection of RNA transcribed from bla-RepB plasmid. Thebla-RepB plasmid expresses a α-lactamase marker in place of the selectorneomycin phosphotransferase. The results with α-lactamase-dependingsystem were in overall agreement with the data obtained exploitingneomycin resistance, taking into account the different sensitivity ofthe systems. Treatment of bla-RepB transfected cB76.1/Huh7 cells withα-IFN reduced the number of blue cells to background levels,demonstrating that the stain was actually depending on RepB replication.

Quantitative analysis of transient replication was performed byreal-time TaqMan. Three days after transfection a 20-30 fold increase ofRepB RNA with respect to a non-replicating control in cB76.1/Huh7 cellswas observed, whereas RepB RNA was below the detection limits inunselected Huh7. The data indicate that the cell giving rise to theclone B76.11Huh7 was capable of sustaining replication at a higherextent than the majority of the other cells in the originallytransfected Huh7 population.

Eight independent cell lines originally transfected with neo-RepB RNAand kept for 2 weeks in the absence of G418 were transfected with RNAtranscribed from the bla-RepB plasmid. All cell lines supportedreplication with an efficiency much higher than that shown by thenon-clonal Huh7 original population (0.005% true blue cells, 3% paleblue cells), ranging from 10 to 80% blue cells, and with a certaindegree of variability in the stain intensity among the various clones.This indicates that most of the originally identified neo-resistantclones were actually originated from the selection of transfected cellsin the total Huh7 population able to enhance replication of GBV-Breplicon RNA with respect to a base-line lying below the detectionthreshold of the selection system used.

Example 8 Use of Enhanced Cells to Achieve Replication of Full-LengthGBV-B

GBV-B full-length genomic FL3 RNA (EMBL accession number AJ277947) wastransfected in Huh7 and in enhanced cells cB76.1/Huh7 in parallel withthe corresponding GAA control. Intracellular GBV-B RNA was measured byTaqman in a time-course experiment.

Replicon enhanced cells were able to support full length genomicreplication. In contrast, transfection of Huh7 with FL3 failed toprovide detectable FL3 replication.

Results of transfection of cB76.1/Huh7 enhanced cells showed that after6 days the amount of FL3 RNA extracted from about 5×10⁵ cells was1.15×10⁶ G.E., whereas the RNA extracted from the same number of cellstransfected with the mutant GAA construct corresponded to 1.15×10⁵ G.E.Treatment of the transfected cells with interferon resulted in a10-20-fold decrease of FL3 RNA amount and did not affect the GAA mutantRNA amount. The lack of sensitivity to interferon of the GAA mutantindicated that the GAA RNA detectable at day 6 post transfectioncorresponded to non-replicated input RNA.

Example 9 Infection of Enhanced Cells with GBV-B

GBV-B virus inoculum produced upon passage of virus in tamarins was usedto infect Huh7 and cB76.1/Huh7 enhanced cells. GBV-B containing tamarinserum was layered onto 10⁵ cells in multiwell-6-wells 1 day afterplating at multiplicity of infection of 0.75 G.E./cell. After 6 hoursadsorption, the virus-containing serum was removed, the cellsextensively washed and incubated in the serum-free DMEM. Cells fromindividual wells were lyzed with Trizol at different times and GBV-B RNAquantified by Taqman.

As an alternative method, 10⁶ cells were mixed with undilutedvirus-containing serum (10⁶ G.E.) and subjected to electroporation. Thecells were plated at a density of 10⁵/well and treated as described forexperiments of RNA electroporation. Duplicate wells were run inparallel, with and without interferon. Cells from individual wells werelyzed with Trizol at different times and GBV-B RNA quantified by Taqman.The amount of G.E. detectable immediately after electroporation and washwas 10³ G.E., it was undetectable at 24 and 48 hours, at day 3 it was10⁴ G.E.; at day 6 it was 5×10³ (possibly inhibition due to celldensity); in corresponding wells treated with interferon the RNA wasundetectable. At day 6 IFN-treated cells were discarded, non-treatedcells were split 1:6 (and duplicates were run +/− interferon), at day 7the RNA raised again to 1 G.E., decreased at almost undetectable levelsat day 10 (inhibition may have been due to cell density). At day 10 thenon-treated cells were split again 1:6. RNA was quantified at day 13 and16 giving a figure corresponding to 4×10³ G.E. and 1.2×10⁴ G.E.respectively. The last point (16 days) value corresponds to about 0.1G.E. per cell. Corresponding points obtained with cells cultivated inthe presence of IFN gave background signals.

Example 10 Production of Additional Replicon Enhanced Cells

Human hepatoma cell lines, HepG2 and Hep3B, were transfected withneo-RepB RNA. Permissive cell lines were observed with Hep3B (obtainedfrom the ATCC), but not with HepG2. Eleven Hep3B permissive cell lineswere further characterized. Hep3B was obtained from the ATCC.

The 11 cell lines show a gradient of permissiveness to GBV-B withrespect to parental Hep3B. The most enhanced is the cell line designated“RepB/Hep3B-9”. A cell line with an intermediate phenotype is“RepB/Hep3B-11”. Mutations were identified for the replicons fromRepB/Hep3B-9 and RepB/Hep3B-11 (Table 2). TABLE 2 Cell line NucleotideGene Amino acid RepB/3B-9 “A” insert in “A” EMCV IRES — stretch1876-1880 A2780G NS3 (hel) Ala296 silent RepB/3B-11 T1827G EMCV IREST2375A NS3(pro) Ser161, silent

Example 11 Production of Replicon Enhanced Cells Supporting A GenomicReplicon

A GBV-B selectable full length replicon (neo-FL-D) was constructed byinserting the genes encoding core-E1-E2-NS2 between EMCV IRES and theGBV-B NS3 in neo-RepD. RNA transcribed from this clone waselectroporated into cB76/Huh7 cells. Three cell lines bearing thefull-length replicon were isolated, the presence and replication ofneo-FL-D was confirmed by qPCR, and normal PCR.

Mutations from three different cell lines supporting neo-FL-D are shownin Table 3. TABLE 3 Cell Line Mut. Gene Nucleotide Amino acidFL-D/Huh7-1.1 NS3 T5222C Ala156, silent FL-D/Huh7-2.1 NS5B A10417GGlu554Gly FL-D/Huh7-3.1 Neo G1050T Arg177Leu Core G2197C Gly88Ala

All three cell lines were cured with IFN. This treatment gave rise tosecond generation cured cells. The second generation cells werere-transfected with neo-RepB, neo-FL-D, bla-FL-D (beta-lactamaseencoding sequence replacing neo in neo-FL-D), and HCV replicons (Con1replicon w.t. sequence and NS5A A227T corresponding mutant). Con 1 isdescribed by Lohmann et al., Science 285:110-113, 1999.

Neo-FL-D and bla-FL-D efficiently replicated only in thesecond-generation-cured cFL/Huh7 cell lines (the best was that producedcuring FL-D/Huh7-1.1 and FL-D/Huh7-2.1). The second-generation-curedcells were not improved compared to parental cB76.1/Huh7 for replicationof the subgenomic neo-RepB replicon. The cells were enhanced for w.tCon1 HCV replicon and, at a higher extent, for NS5A A227T mutant HCVreplicon compared to cB76.1/Huh7 (the best was that produced curingFL-D/Huh7-3.1).

Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1. A GBV-B replicon comprising the following regions: a GBV-B 5′ UTRsubstantially similar to bases 1-445 of SEQ ID NO 1; a selection orreporter sequence functionally coupled to said GBV-B 5′ UTR; an internalribosome entry site; a NS3-NS5B sequence substantially similar to bases1938-7709 of SEQ ID NO: 1 functionally coupled to said internal ribosomeentry site and an AUG translation initiation codon; and a GBV-B 3′ UTRsubstantially similar to bases 7710-8069 of SEQ ID NO: 1, wherein saidreplicon is capable of replication in a cell.
 2. The GBV-B replicon ofclaim 1, further comprising a GBV-B structural region, wherein saidGBV-B structural region is functionally coupled to said GBV-B 5′UTR. 3.The GBV-B replicon of claim 2, wherein said GBV-B structural regioncomprises a sequence substantially similar to a sequence selected fromthe group consisting of: bases 446-511 of SEQ ID NO: 1, bases 446-487 ofSEQ ID NO: 1, bases of 446-469 of SEQ ID NO: 1, the RNA version of bases446-2641 of SEQ ID NO: 2, and the RNA version of bases 446-3265 of SEQID NO:
 2. 4. The GBV-B replicon of claim 3, wherein said repliconconsists of: said GBV-B 5′UTR; said GBV-B structural region; saidselection or reporter sequence; said internal ribosome entry site; saidNS3-NS5B sequence; and said GBV-B 3′ UTR.
 5. The GBV-B replicon of claim4, wherein said internal ribosome entry site has the sequence of bases1324-1934 of SEQ ID NO 1; said GBV-B structural region consisting of asequence selected from the group consisting of: bases 446-511 of SEQ IDNO: 1, bases 446-487 of SEQ ID NO: 1, bases of 446-469 of SEQ ID NO 1,the RNA version of bases 446-2642 of SEQ ID NO: 2 and the RNA version ofbases 446-3265 of SEQ ID NO: 2; said NS3-NS5B region is Met-NS3-NS5Bregion consisting of bases 1935-7709 of SEQ ID NO: 1; and said GBV-B 3′UTR is bases 7710-8069 of SEQ ID NO:
 1. 6. The GBV-B replicon of claim5, wherein said GBV-B structural region consists either of the RNAversion of bases 446-2642 of SEQ ID NO: 2 or the RNA version of bases446-3265 of SEQ ID NO:
 2. 7. The GBV-B replicon of claim 1, wherein saidreplicon consists of SEQ ID NO:
 1. 8. The GBV-B replicon of claim 2,wherein said GBV-B structural region comprises a sequence substantiallysimilar to a sequence selected from the group consisting of: bases446-511 of SEQ ID NO: 1, bases 446-487 of SEQ ID NO: 1, bases of 446-469of SEQ ID NO: 1, and the RNA version of bases 446-2641 of SEQ ID NO: 2.9. The GBV-B replicon of claim 3, wherein said replicon consists of:said GBV-B 5′ UTR; said selection or reporter sequence; said internalribosome entry site; said GBV-B structural region; a NS2-NS5B regioncomprising a NS2 region substantially similar to the RNA version ofbases 2642-3265 of SEQ ID NO: 2 joined to the 5′ end of said NS3-NS5Bregion; and said GBV-B 3′ UTR.
 10. The GBV-B replicon of claim 9,wherein said internal ribosome entry site has the sequence of 1324-1934of SEQ ID NO 1; said GBV-B structural region comprises a sequenceselected from the group consisting of: bases 446-511 of SEQ ID NO 1,bases 446-487 of SEQ ID NO 1, bases of 446-469 of SEQ ID NO 1, and theRNA version of bases 446-2641 of SEQ ID NO: 2; said NS2-NS5B is aMet-NS2-NS5B region consisting of said 5′ AUG translation initiationcodon, said NS2 region, and said NS3-NS5B region, wherein said NS2region consists of the RNA version of bases 2642-3265 of SEQ ID NO: 2and said NS3-NS5B consists of bases 1938-7709 of SEQ ID NO: 1; and saidGBV-B 3′ UTR is bases 7710-8069 of SEQ ID NO:
 1. 11. The GBV-B repliconof claim 10, wherein said replicon produces an infectious virion.
 12. Anexpression vector comprising a promoter transcriptionally coupled to anucleotide sequence coding the GBV-B replicon of claim
 1. 13. A GBV-Breplicon made by a process comprising the steps of transfecting a cellwith the replicon of claim 1 and isolating said replicon.
 14. The GBV-Breplicon of claim 13, wherein said cell is either a Huh7 cell, a Hep3Bcell, is derived from a Huh7 cell, or is derived from a Hep3B cell. 15.A method of making a second GBV-B replicon from a first GBV-B repliconcomprising the steps of: a) transfecting a cell with said firstreplicon, wherein said first replicon is the replicon of claim 1; b)isolating a replicon from said transfected cell; c) determining thenucleotide sequence of said replicon from said transfected cell; and d)producing said second replicon, wherein said second replicon containsthe first replicon sequence with one or more alterations correspondingto said replicon from said transfected cell.
 16. The method of claim 15,wherein said cell is either a Huh7 cell, a Hep3B cell, is derived from aHuh7 cell, or is derived from a Hep3B cell.
 17. A method of measuringthe ability of a compound to affect GBV-B replicon activity comprisingthe steps of: a) providing said compound to a cell containing the GBV-Breplicon of claim 1; and b) measuring the ability of said compound toaffect one or more replicon activities as a measure of the effect onGBV-B replicon activity.
 18. The method of claim 17, wherein said cellis a human hepatoma cell.
 19. The method of claim 18, wherein said saidcell is either a Huh7 cell, a Hep3B cell, is derived from a Huh7 cell,or is derived from a Hep3B cell.
 20. A GBV-B replicon enhanced cell,wherein said cell has an maintenance and activity efficiency of at least25% when transfected with a GBV-B replicon of SEQ ID NO: 1 by theElectroporation Method. 21-23. (canceled)
 24. A method of making a GBV-Breplicon enhanced cell comprising the steps of: a) introducing andmaintaining the GBV-B replicon of claim 1 in a cell; and b) curing saidcell of said GBV-B replicon to produce said replicon enhanced cell.25-26. (canceled)
 27. A method of making a GBV-B replicon enhanced cellcontaining a functional GBV-B replicon comprising the steps of: a)introducing and maintaining a first GBV-B replicon in a cell, whereinsaid first replicon is the replicon of claim 1; b) curing said cell ofsaid first replicon to produce a cured cell; and c) introducing andmaintaining a second GBV-B replicon into said cured cell, wherein saidsecond GBV-B replicon may be the same or different than said first GBV-Breplicon. 28-47. (canceled)